A central problem in neurobiology is to determine how the complex networks of neuronal connections, that underlie the functioning of the nervous system, are established. This intricate pattern is generated, at least in part, when neuronal cell bodies and their axons migrate to their targets, guided by diffusible chemotropic cues. The long term goal for my laboratory is to understand how cells detect and orient themselves in gradients of guidance cues, and transduce this information intracellularly into changes in shape and motility that lead to directed migrations. Netrin-1 is an example of such a guidance cue, and two families of receptors that specify attractant and repellent responses to netrin-1 have been identified. The attraction of a cell or axon toward netrin-1 is mediated by DCC (Deleted in Colorectal Carcinoma). Studies in C. elegans demonstrate that repulsion away from netrin-1 is specified by UNC5 receptors, and that UNC5 functions together with DCC to mediate this repulsion. In the preliminary data, it is shown that UNC5H and DCC physically interact to form a repellent protein complex. Based on these studies, the major hypothesis of the grant proposal is that vertebrate homologs of UNC5 (UNC5Hs), together with DCC, mediate netrin-1 dependent chemorepulsion in vertebrates in vivo. To address the hypothesis, the specific aims of this proposal are two-fold. I) To define the in vivo role of the UNC5H/DCC complex in directing netrin-1 mediated repulsion. Studies are proposed on the role of netrin-1 in guiding the migration of cerebellar granule cells. These studies examine the consequences of netrin- 1 loss-of-function and gain-of-function in vivo, and the migration of cerebellar granule cells in vitro. II) To understand the molecular mechanisms that specify repulsion versus attraction, the molecules that bind to UNC5Hs and transduce a repellent signal will be identified using yeast two-hybrid and affinity chromatography approaches. The function(s) of these interacting proteins in mediating repulsion in response to netrin-1 will be tested in axon guidance or cell migration assays. These experiments provide the foundation for understanding how cells translate a netrin-1 signal into directed migrations.